Durchflußzytometrische Charakterisierung von Reifungsvorgängen und stammzellnahen Zellpopulationen bei der B-Vorläuferzell-ALL im Kindesalter

Abstract

Bone marrow and peripheral blood from children with acute lymphoblastic leukemia was analyzed by flow cytometry to assess leukemic cell differentiation and to characterize the profile of cell surface marker expression on rare CD34+ cell populations. The goal of this study was to determine if patterns of cell surface antigens could be identified on CD34+ subpopulations which may allow distinction between normal and leukemic stem cells. Expression of the progenitor cell antigen CD34 on leukemic blasts was very heterogeneous and varied between 0.5 and 100% in 20 patients analyzed in this study. In cALL and pre-B-ALL, a variable percentage of the leukemic cells coexpressed CD20 in addition to CD10. Only in one case, differentiation characteristic for normal B cell development with coordinated downregulation of CD10 with increasing expression of CD20 was observed. By analysing 5 x 10(6)-1 x 10(6) cells, a CD34+ cell population could be identified in 8 out of 8 patients which did not express CD19 and comprised less than 0.1% of all bone marrow or peripheral blood cells. Within this population, there was differentiation from primitive CD34-CD38- to more mature CD34+CD38+ cells. In 4 of these patients, an additional CD34+ population with low expression of CD19 (CD34+CD19lo) was detected. The lack of CD45 expression on the leukemic cells of 2 patients was used as a marker for the leukemic cell clone. In both patients, the CD34+CD19- cells did express CD45 while CD34+CD19lo/+ cells were CD45 negative. This suggests that the CD34+CD19lo cells were part of the leukemic clone and that the CD34+CD38-CD19- cells may represent residual normal primitive hematopoietic cells. In conclusion, flow cytometry allowed identification of primitive CD34+ cell populations in children with ALL, which can now be functionally characterized by transplantation onto immune-deficient mice.

Expression of AC133 and CD117 on candidate normal stem cell populations in childhood B‐cell precursor acute lymphoblastic leukaemia

“…There was no correlation between the expression of AC133 and CD34 within the leukaemic cell clone. Moreover, and in accordance with what has recently been described in AML (Kratz-Albers et al, 1998), an asynchronous expression of AC133 on CD34-negative leukaemic cells was observed in eight patients (Baersch et al, 2000).…”

“…The immature CD34 + CD19 − cell compartment was analysed by four‐colour flow cytometry as previously described ( Baersch et al , 1996 ; Vormoor et al , 1998 ). Briefly, 5 × 10 6 unseparated or ficolled bone marrow cells were incubated for 20 min at room temperature with saturating amounts of the following antibodies: anti‐CD34‐FITC/anti‐AC133‐PE/anti‐CD45‐PerCP/anti‐CD19‐APC (581, Immunotech/AC133, Miltenyi Biotec GmbH/HLe‐1, Becton Dickinson Immunocytometry Systems (BDIS)/SJ25C1, BDIS) or anti‐CD34‐FITC/anti‐CD117‐PE/anti‐CD45‐PerCP/anti‐CD19‐APC (581, Immunotech/95C3, Immunotech/HLe‐1, BDIS/SJ25C1, BDIS).…”

Flow cytometric identification of candidate normal stem cell populations in CD45‐negative B‐cell precursor acute lymphoblastic leukaemia

“…For the following analysis, a flow cytometric protocol was established to characterize rare CD34 + CD19 − cells in the bone marrow of ALL patients ( Baersch et al , 1996 ). This was achieved by increasing the number of events analysed to 1 × 10 6 .…”

“…Büchner for providing normal bone marrow samples and PD Dr B. Dockhorn‐Dworniczak, Dr S. Könemann and Dr M. Zühlsdorf for critically reviewing the manuscript. Flow cytometric data of patient 7 have been described before ( Baersch et al , 1996 ). Data were presented in part at the international symposium Acute Leukemias VII, 22–26 February 1997, Münster, Germany ( Annals of Hematology , 74, (Suppl.…”

“…In AML, cells with a CD34 + CD38 − stem cell phenotype may be rare (< 1%) ( Terstappen et al , 1992 ), nevertheless only leukaemic cells with this phenotype have been shown to possess stem cell properties and are able to regrow the leukaemia in immunodeficient mice ( Bonnet & Dick, 1997; Lapidot et al , 1994 ). Therefore a flow cytometric protocol was developed to characterize rare immature non‐B‐lymphoid CD34 + CD19 − cells in the bone marrow and peripheral blood of patients with B‐cell precursor ALL ( Baersch et al , 1996 ).…”